DESCRIPTION (Applicant's Description Verbatim): Our long-term goal is to elucidate the molecular and cellular events underlying the initiation of hematopoiesis in the mammalian embryo. During development, primitive eiythroblasts arise in the yolk sac while definitive blood cells subsequently differentiate in the fetal liver and bone marrow. While primitive and then definitive hematopoietic progenitors first arise in the mouse yolk sac during gastrulation (E7.O-E8.5), hematopoietic stem cells (HSCs) with the ability to engraft adult recipients are not found in the embryo until ElO. M. Yoder (Indianapolis) has recently identified novel "embryonic about HSCs at E9.O that provide longterm engraftment ol newborn, but not adult, recipients. We hypothesize that these embryonic HSCs arise from yolk sac mesoderm cells during gastrulation and subsequently migrate to the fetal liver. In the first aim of this proposal, we will use this newborn transplantation system in collaboration with M. Yoder to determine the developmental origin and intraembryonic sites of migration of mamMALIAN embryonic HSCs. Bone marrow cells from primary recipients of embryonic HSCs can engraft secondary adult recipients. This indicates that E9.O embryonic (fetal liver homing) HSCs can become "adult" (bone marrow homing) HSCs. We hypothesize that this transition requires the differential expression of molecules associated with cell adhesion and migration, such as selectins, integrins, Ig-like proteins, and chemokines. In the second aim of this proposal, subtractive hybridization will be used to define the molecular differences between embryonic and adult HSCs. We will use both commercial and proprietary cDNA arrays with bioinfornatic analysis, as well as PCR-based screens, to focus on differences in molecules associated with cell adhesion and migration. In the third aim, we will investigate the function of candidate molecules in the transition from embryonic to adult HSC. Primary embryonic HSCs transduced with differentially expressed molecules will be screened for the ability to function as adult HSC both in vitro (cobblestone assay) and subsequently in vivo (transplant of adult recipients). A better understanding of the initiation of mammalian hematopoiesis will provide insights into the ontogeny, regulation and expansion of HSCs, as well as the origin of leukemias and bone marrow failure syndromes.